富含小鼠骨髓间充质干细胞的生物活性小鼠烧伤变性脱细胞真皮基质的制备

Preparation of bioactive denatured acellular dermal matrix from burn mice riched in mice bone marrow mesenchymal stem cells

目的探讨富含小鼠骨髓间充质干细胞的生物活性小鼠烧伤变性脱细胞真皮基质(DADM)的制备。方法取12只BALB/c小鼠,背部皮肤造成20%体表总面积深Ⅱ度烫伤(以下称烧伤)。取烧伤皮肤,去除表皮后,按随机数字表法分为聚乙二醇辛基苯基醚(Triton X-100)组和乙二胺四乙酸(EDTA)组,每组15个样本。Triton X-100组样本置于2.5g/L Triton X-100-2.5g/L胰蛋白酶混合溶液中持续振荡洗脱24h,EDTA组样本置于0.2g/L EDTA-2.5g/L胰蛋白酶混合溶液中持续振荡洗脱24h制备小鼠DADM。对DADM行大体观察,扫描电镜下观察DADM胶原纤维的结构及排列,荧光显微镜下观察DADM的组织结构。将小鼠骨髓间充质干细胞(BMSC)以每孔2×10^5个的浓度接种于2组小鼠DADM中,制备成具有生物活性的小鼠DADM。培养3d,苏木素-伊红染色观察具有生物活性的小鼠DADM的组织结构,免疫荧光法观察具有生物活性的小鼠DADM中BMSC的分布及数目;细胞计数试剂盒8检测培养2h、1d、3d、5d具有生物活性的小鼠DADM中BMSC的增殖情况。对数据行重复测量方差分析及t检验。结果(1)2组小鼠DADM外观均呈白色,具有一定的韧性及弹性。2组小鼠DADM呈良好的三维多孔网状结构,EDTA组小鼠DADM胶原纤维连续性良好,Triton X-100组小鼠DADM胶原纤维发生不同程度断裂。2组小鼠DADM均洗脱完全,胶原纤维疏松、排列紊乱,EDTA组小鼠DADM组织结构连续性较Triton X-100组佳。(2)培养3d,2组具有生物活性的小鼠DADM中BMSC均分布均匀,EDTA组具有生物活性的小鼠DADM中BMSc为(37±7)个,显著高于Triton X-100组的(25±8)个,t=0.128,P<0.05。培养2h、1d,Triton x-100组和EDTA组具有生物活性的小鼠DADM中BMSC增殖能力相近(t=1.292、0.656,P>0.05);培养3、5d,EDTA组具有生物活性的小鼠DADM中BMSC增殖能力显著高于Triton X-100组(t=2.309、14.128,P<0.05或P<0.01)。结论EDTA洗脱制备的小鼠DADM具有更佳的三维多孔网状结构,胶原纤维连续性好;移植BMSC制备的具有生物活性的小鼠烧伤DADM中BMSC分布均匀、数量多、增殖能力强,有望成为较佳的自体真皮替代物。

Objective To investigate the preparation of bioactive denatured accllular dermal matrix (DADM)from burn mice riched in mice bone marrow mesenchymal stem cells.Methods Twelve BALB/c mice were collected and 20%total body surface area scalds (hereinafter referred to as burns)with deep partial thickness were inflicted on the back skin of each mouse.After removing epidermis,the burned skin were collected and divided into Triton X-100group and elhylcne diamine tetraacetic acid (EDTA) group according to the random number table,with 15samples in each group.Samples in Triton X-100 group and EDTA group were respectively placed in mixture of 2.5g/L Triton X-100 and 2.5g/L trypsin solution and mixture of 0.2g/L EDTA and 2.5g/L trypsin solution for sustained vibration and elution for 24hours to make mice DADM.The general appearance of DADM was observed.The structure and arrangement of eolla-gen fibers of DADM were observed by scanning electron microscope and tissue structure of DADM were observed by fluorescence microscope.Bone marrow mesenchymal stem cells (BMSCs)from mice were trans-planted in mice DADM in the two groups with concentration of 2×105 ccells per well to prepare bioactive mice DADM.After cultured for 3days,tissue structure of bioactive mice DADM was observed by hematoxy- 1in and eosin staining,distribution and number of BMSCs of bioactive mice DADM were observed by immu- nofluorescence staining.Proliferation of BMSCs of bioactive mice DADM after cultured for 2h,1d,3d, and 5d was detected by cell count kit-8.Data were processed with analysis of variance for repeated measurement and t test.Results (1)Mice DADM in the two groups were white in appearance with certain tenaci- ty and elasticity.Mice DADM in the two groups maintained good three-dimensional porous network structure. Collagen fibers of mice DADM in EDTA group were with good continuity,and collagen fibers of mice DADM in Triton X-100group were fractured in varying degrees.Mice DADM in the two groups were deeellularized completely,and the collagen fibers were loose and arranged disorderly.The continuity of tissue structure of mice DADM in EDTA group was better than that of mice DADM in Triton X-100group.(2)After cultured for 3days,the BMSCs in bioactive mice DADM in the two groups were evenly distributed.The number of bioactive BMSCs in mice DADM in EDTA group was 37±7,which was significantly more than that of mice DADM in Triton X-100group (25±8,t =0.128,P <0.05).The proliferation of bioactive BMSCs in mice DADM in Triton X-100group and EDTA group was similar at 2hours and on day 1after euhured (t = 1.292,0.656,P >0.05).On 3,5days after cultured,the proliferation of bioactive BMSCs in mice DADM in EDTA group was significantly higher than that of mice DADM in Triton X-100group (t =2.309, 14.128,P <0.05or P <0.01).Conclusions Mice DADM prepared by decellularization of EDTA has better three-dimensional porous network structure and good continuity of collagen fiber.The BMSCs in bioactive DADM from burn mice prepared by transplanting BMSCs are evenly distributed with large quantity and strong proliferative capacity,which has the potential to be good autologous dermal substitute.